Membrane receptor reagent and assay

ABSTRACT

A membrane receptor reagent and assay is disclosed in which liposomes are bound to an evanescent wave emitting surface. Membrane receptors on the liposome&#39;s fluid lipid bilayer membrane are labeled with a fluorescent or luminescent moiety. These membrane receptors are free to diffuse randomly throughout the liposome surface, and thus tend to redistribute according to externally applied forces. The evanescent wave-emitting surface additionally contains reagents that reversibly bind to the membrane receptors, tending to bring them closer to region of high evanescent wave intensity. Test analytes that disrupt or promote the association between the membrane receptors and the surface reagents act to change the average distance between the membrane receptors and the evanescent wave emitting surface, resulting in a change in the fluorescent or luminescent signal. This reagent and assay system functions with physiologically important membrane receptors such as GPCR receptors, other 7-tm receptors, drug transport proteins, cytochrome P450 membrane proteins and other clinically important membrane components. The reagent and assay methods may be incorporated into microarrays, capillaries, flow cells and other devices, and used for drug discovery, ADMET, and other biomedically important assays.

[0001] This patent is a continuation in part of patent application Ser.No. 10/444,390 “Membrane receptor reagent and assay”, filed May 23,2003. Application Ser. No. 10/444,390 claimed priority benefit ofprovisional patent applications 60/389,679; and 60/400,396 both entitled“Tethered receptor-ligand reagent and assay”, filed Jun. 17, 2002 andJul. 31, 2002; and provisional patent application 60/428,137; entitled“Membrane receptor reagent and assay”, filed Nov. 21, 2002.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention generally concerns reagents and methods useful formembrane receptor-ligand binding assays.

[0004] 2. Description of the related art

[0005] Much of modern pharmacology and biochemistry is focused on theinteractions between different types of biological signaling moleculesand their corresponding membrane receptors. Structurally, these membranereceptors are often G-Protein Coupled Receptors (GPCR) from the7-transmembrane (7tm) protein family, and other types of related7-transmembrane proteins such as ion channels.

[0006] Biological signaling molecules and membrane receptors are presentin many physiological processes, and are particularly important fornervous system function. Indeed, nervous system membrane receptorsystems, such as the dopamine, serotonin, and opioid receptor families,have been found to be involved in many mental disorders, such asanxiety, depression, and drug abuse. Furthermore, these membranereceptors have been found to be excellent drug targets. GPCR reactivedrugs are involved in many other biological processes as well. Kenakin,Annu Rev Pharmacol Toxicol 2002;42:349-79),.

[0007] To date, however, only a small number of the many possibleinteractions between the millions of potential candidate drug ligands,and the thousands of different membrane receptors, have been wellcharacterized. With the recent advances in both genomics and nucleicacid microarray technology, the cellular distribution and sequence ofthese membrane receptors and receptor families are now readilyavailable. As a result, one of the key challenges going forward is toutilize this new knowledge to aid in the development of next generationdrugs.

[0008] Modern drug discovery and development is a multi-step process.Usually, one or more medically important target receptors areidentified, a large number of prototype lead drugs are synthesized, andappropriate High Throughput Screening (HTS) assays are conducted toassess the proper differential binding to an initial set of selectedtarget and non-target receptors. Those lead candidates that survive theprocess are then subjected to progressively more expensive and stringentassays; whole cell assays, animal studies, Absorption, Distribution,Metabolism, Excretion, Toxicity (ADMET) studies, and finally human PhaseI, II, and III studies. The expense and time involved in the laterstages, typically hundreds of millions of dollars and many years, aresuch that it is enormously important that optimal leads be found asearly in the process as possible.

[0009] One of the best ways to exploit the recent advances in genomicsis to use the information to clone and express these membrane receptorsin a pure state, and use these cloned receptors for HTS leadidentification and optimization. In theory, the later stages of drugdevelopment could be significantly streamlined if low-cost and efficientHTS methods were developed to initially optimize a lead's specificbinding to its target receptor, and to detect any unwantedcross-reaction with non-target receptors.

[0010] At present, however, membrane HTS screening methods aresub-optimal. Membrane proteins are hydrophobic, and typically onlyassume their correct physiological conformation in a lipid bilayermembrane environment. Moreover, many membrane receptor proteins relyupon certain aspects of lipid bilayers, such as receptor lateralmobility, association with lipid rafts, association with other proteins,small molecule cofactors etc., for proper function. Such faithfulrecreations of native membrane structures are difficult to reproduce ina synthetic, in-vitro, environment.

[0011] As a result, present membrane HTS screening methods usually relyupon the binding of radioactively labeled ligands to natural membranereceptors (e.g. receptors obtained from natural sources such as culturedcells). These natural membrane receptors are often bound to a solidphase, such as a filter or microwell plate. Radioactive ligand isapplied to the sample, followed by one or more washing steps. The boundradioactive ligand is then detected by its radioactive scintillationsignal. Thus relatively large quantities of membrane proteins andcandidate drug ligands are required for each assay. For example, eventhe optimized Packard Bioscience FlashPlate™ system requires 25 to 50 ulof fluid for each assay point.

[0012] Although the use of natural membranes has strong physiologicalmerit, it is slow, expensive, and cumbersome. An alternate membranereceptor HTS methodology that could return physiologically useful datawith cloned membrane receptors would be highly advantageous, as it wouldenable the many recent genomic insights to be easily and rapidly used.

[0013] An ideal membrane-receptor HTS methodology would have a number ofother characteristics as well. At present, HTS methods are typicallyrestricted to large, well-financed, commercial organizations. This isbecause the present methodologies require the use of large quantities ofexpensive membrane receptors, expensive synthetic drug candidates, andexpensive automation. If alternative methods could be devised to reducethe quantities of receptors and synthetic drug candidates by severalorders of magnitude, the financial and logistical burden of HTS studieswould be greatly eased. This would enable a much larger number ofreceptors and drug candidates to be screened, and could also make HTSmethods feasible in smaller scale settings, such as academiclaboratories. One good way to accomplish this goal is by the developmentof suitable membrane receptor microarray technology.

[0014] Microarray technology: In recent years, microarrays have becomewidely used for genomic and proteomic biotechnology, biomedicalresearch, and biomedical diagnosis. In particular, microarray methodshave become widely used for nucleic acid research, and a large number ofnucleic acid microarrays are commercially available from AffymetrixInc., Incyte Pharmaceuticals, Inc., and many other companies. Thesemethods (reviewed in Schena, Microarray Biochip Technology (2000) EatonPublishing, Natick, Mass.) generally work by binding a large number ofnucleic acid microsamples to the surface of a flat support. Samplescontaining one or more unknown complementary nucleic acids are thenexposed to the nucleic acid microarray, and the sample is allowed tohybridize to the microarray. Hybridized nucleic acids are then detectedby various means, and the overall nucleic acid composition of theunknown sample is assessed.

[0015] The general principle has been that to detect two biologicallyinteracting elements that form a pair, such as complementary nucleicacid strands, the microarray will contain one-half of the pair, theunknown analyte will contain the other half of the pair, and theinteraction between the two elements will generate a detectable signal.

[0016] A good overview of protein microarray technology in general, asit exists at the time of this patent application, can be found in thearticle by Mitchell, “A perspective on protein microarrays”, Naturebiotechnology (20), 2002, 225-229, the contents of which areincorporated herein by reference.

[0017] There have been some previous attempts to produce membranereceptor microarrays, such as U.S. Pat. No. 6,228,326; and Salafsky et.al., Biochemistry (1996) 35: 14773-14781. One approach, pioneered byBoxer and coworkers (Groves et. al., Science (1997), 275: 651-653)relies upon the formation of an artificial planar lipid membraneparallel to a microarray surface. The microarray uses a series ofmechanical barriers to separate one lipid region from another, enablingmultiple regions to be patterned. The microarray is analyzed byfluorescence recovery after photobleaching (FRAP) techniques. Here,fluorescent membrane components are exposed to a localized region ofintense laser irradiation. Following irradiation, a bleached regionforms, which can be observed by a fluorescence microscope. Thisgradually disappears as unbleached membrane components from surroundingregions gradually migrate into the bleached region.

[0018] Although useful for demonstration purposes, one fundamentalcritique of this approach is that due to the close (1 nm) associationbetween the inner leaflet of the artificial membrane lipid bilayer, andthe solid phase support, the physiology of the artificial membrane ishighly distorted. In particular, larger membrane proteins, such as GPCRproteins, are large enough that they can interact with the solid phasesupport, resulting in distorted conformations and inactivity.Additionally, the narrow aqueous layer is too thin to enable GPCRproteins to interact with other cytoplasmic side cofactors, which play akey role in the proper function of the receptor.

[0019] A second critique of this approach is that FRAP detectiontechniques are too crude to spot many interesting types of interactions.For example, these methods are of marginal utility for drug discoverypurposes. This is because the binding of the relatively small (lowmolecular weight) drug candidates to relatively large (high molecularweight) membrane receptors will have a negligible impact on the FRAPmobility of the receptors, and thus will be poorly detected by FRAPtechniques.

[0020] An alternative type of membrane microarray has been proposed byZiauddin and Sabatini that relies on DNA uptake (Ziauddin J, Sabatini DM, “Microarrays of cells expressing defined cDNAs”, Nature, 2001,411:107-10). Here, DNA coding for receptor genes is spotted on amicroarray surface. After spotting, cells are cultured on themicroarray. Those cells located above the DNA spots take up the DNA, andexpress the appropriate receptor on the cell surface. This is arelatively new approach, and more work will be needed before therelative utility of such microarrays can be accurately assessed.

[0021] The “L1” chip produced by Biacore Corporation represents a thirdtechnique (Williams, C., Cook S., Knoppers M., “Advances in LipidImmobilization: The L1 Chip”, BIAjournal 2000, 7 (1)). In thistechnique, membrane fragments from mammalian cells are bound to thesurface of a flow-cell. Samples containing potential ligands areinjected into the flow cell, and after a washing step, retained materialis eluted and analyzed by mass spectroscopy.

[0022] Although this technique is clearly useful for certain analyticalneeds, it has some drawbacks. The present L1 chip design has hydrophobicmoieties on the surface of the chip. These interact with membranesamples, causing liposomes and membrane vesicles to rupture. As aresult, the applied membranes become firmly attached to the surface ofthe chip with no aqueous separation layer (Erb E, Chen X, Allen S,Roberts C, Tendler S, Davies M, Forsen S. “Characterization of thesurfaces generated by liposome binding to the modified dextran matrix ofa surface plasmon resonance sensor chip” Anal Biochem 2000,280(1):29-35). Thus, as is the case for the Boxer design, ligand-bindingreactions are distorted. Additional drawbacks are the relatively slowthroughput of the single element flow-cell design, and a large number offalse positives produced by the mass-spectrometer detection methodology.

[0023] Evanescent wave technology: Evanescent waves are generated when alight wave undergoes total internal reflection at a surface (a junctionbetween two media with different indexes of refraction). A light wavetravels through the first media until it encounters the surfaceboundary, and, at the appropriate angle of incidence (the criticalangle), bounces back from the surface and continues traveling throughthe first media, rather than entering into the second media. Here, asmall amount of energy, termed an evanescent wave, penetrates anextremely short distance into the second media, and rapidly decays inintensity as distance from the boundary increases. For opticalfrequencies and media types commonly used in biological research, theevanescent wave decreases in intensity by about 1/e (that is, about1/2.71) over a distance of about 260 nm. Thus, over distances of a fewhundred nanometers, evanescent wave excitation is a good way todetermine the relative distance between an excitable moiety, and asurface boundary.

[0024] Evanescence biosensor techniques are known in the art, but havenot been generally used for membrane bound analytes. For example, U.S.Pat. No. 6,316,274 teaches multi-analyte biosensor methods usingfluorescence moieties excited by evanescence illumination. Otherrepresentative prior art includes U.S. Pat. Nos. 6,274,872; 5,512,492;and 6,395,558.

[0025] Lipid membrane and liposome technology: As first described bySinger and Nicolson (Science 1972 Feb. 18; 175(23):720-31), biologicalmembranes are composed of a hydrophobic lipid bilayer, with membraneproteins existing as compact “ice berg” like structures floatingembedded in this lipid bilayer, which is about 6 nm thick. Due to theweak nature of hydrophobic bonds, lipids and integral membrane proteinsare able to move freely (lateral mobility) within the plane of the lipidmembrane. In particular, when exposed to forces from external ligands,such as bivalent antibodies, lectins, etc., membrane proteins and otherlipid components are able to diffuse together to form “patches” or“caps” on the membrane surface.

[0026] Because biological lipid membranes are highly complex structureswith many different types of membrane proteins, biochemists andmolecular biologists typically find it preferable to work withsimplified synthetic lipid membranes, reconstituted from purifiedcomponents. Such synthetic membranes can be synthesized by simplemethods, since they tend to spontaneously form when lipid mixtures(which may also contain membrane proteins) are dissolved in detergents,and the detergent then gradually removed by dialysis or other process.The resulting synthetic membrane structure typically forms as a“soap-bubble-like” structure called a “liposome” or “phospholipidvesicle”. The walls of the liposome consist of lipid bilayers, and themembrane proteins typically insert themselves into the artificial lipidbilayer with the correct, or nearly correct, conformation. Dependingupon the synthetic process, liposomes can be formed with diametersranging from about 50 nm to 5,000+ nm.

[0027] Artificial liposomes (phospholipid vesicles) containing lipidsand protein can be created by a number of methods, including cadmiumsynthesis (Thromb Haemost 1980 Aug. 29; 44(1): 12-5); dialysis againstoctyl glucoside (Biochemistry 1986 Jul. 15; 25(14): 4007-20);deoxycholate (Biochem J 1977 Jul. 1; 165(1): 89-96), and numerous othermethods.

[0028] Membrane proteins: Many biologically relevant proteins aretransmembrane proteins. These proteins exist embedded in membrane lipidbilayers, and typically can best be studied while associated with intactmembranes. These transmembrane proteins include the 7-transmembraneproteins (reviewed by Kilpatrick et. al. J Cell Sci 2001 February;114(Pt4):629-41), and their medically relevant subfamily, the G-protein cellreceptors (GPCR family), (reviewed by Woodside, Sci STKE 2002 Mar.19;2002(124):PE14). Other relevant transmembrane proteins include theintegrin family, the cadherin family (reviewed by Angst, et. al, J CellSci 2001 February;114(Pt 4):629-41), and many others.

[0029] ADMET assays: The current art in in-vitro ADMET assays isdescribed by Darvas and Dorman in High-Throughput ADMETox Estimation: InVitro & In Silico Approaches, (2002) BioTechniques Press, EatonPublishing, Westborough, Mass.

[0030] An ideal membrane receptor microarray would be bothphysiologically realistic and biochemically well defined. To do this, anideal microarray should present membrane targets in an environment that,from both sides of the lipid bilayer, enables receptor binding proteinsand other cofactors to interact in a normal manner. Additionally, anideal membrane microarray should be able to detect the binding reactionsbetween large numbers of different low molecular weight drug candidateligands, and different membrane receptors. Finally, an ideal membranereceptor microarray should function using ultra-small quantities ofcandidate drug ligands, and ideally also be reusable.

SUMMARY OF THE INVENTION

[0031] Here, a novel type of membrane-receptor reagent, suitable forhigh performance membrane receptor microarrays, is taught. As will bediscussed in later sections, this type of microarray may have highutility for HTS drug discovery and development. For example, acomprehensive GPCR microarray, containing a large number of differentGPCR families and common GPCR variants, would detect unwantedcross-reactions at the earliest stage of the drug discovery process.This would greatly facilitate the development of highly specific andwell-targeted new drugs.

[0032] The invention consists of a reagent system for monitoring theinteraction between one or more membrane receptors of interest (heredesignated as “target membrane receptors”), and one or more experimentalligands. These experimental ligands may be drugs, drug candidates,receptor agonists, antagonists, inhibitors, ect., and are heredesignated as “test ligands” This reagent system consists of:

[0033] (1) A fluid lipid membrane containing a target membrane receptormolecule labeled with a moiety that produces a detectable signal uponreceiving excitation energy, and in which the fluid lipid membrane actsas a flexible tether for the receptor.

[0034] (2) A “reagent ligand” tethered to a surface that emits energycapable of exciting the receptor's detectible signal emitting moiety.This excitation energy diminishes sharply in intensity as distance fromthe surface increases. This reagent ligand binds to the target membranereceptor molecule in a reversible manner. This reagent ligand is alwayspresent in the reagent portion of the system, and it's binding to thetarget membrane receptors may, or may not, be disrupted depending uponthe presence and properties of the unknown “test ligand”.

[0035] (3) Anchor linking means that bind the lipid membrane and thesurface into a single linked structure. This structure utilizes thefluid nature of the lipid membrane to allow the target membranereceptors to associate and dissociate from their binding site on thereagent ligands. The geometry and the tethering action of the fluidlipid membrane is such that if the bond between the target membranereceptor and the reagent ligand is disrupted, the target membranereceptor is free to diffuse far enough away from the energy emittingsurface so as to produce a significant change in the amount ofexcitation energy received by the moiety associated with the targetmembrane receptor.

[0036] Typically, the lipid membrane is in the form of a liposome. Inuse, the reagent system is typically exposed to one or more testligands. These test ligands may either enhance or disrupt theassociation between the target membrane receptor and the surface boundreagent ligand. The ability of the test ligand to modulate thisassociation is thus monitored by observing changes in the detectablesignal emitted by the moiety labeled target membrane receptor.

[0037] Usually, the liposome-associated target membrane receptormolecule will be a transmembrane protein that is of biological interest,such as the 7-transmembrane proteins (particularly “GPCR” proteins),toll-like receptors, transport proteins, biological response modifierreceptor proteins, coagulation factors, immune response receptors,enzymes, and other biologically interesting cellular receptors.

[0038] The reagent ligand that is tethered to the surface may be anymolecular entity that binds to the liposome-associated target membranereceptor molecule. Usually, the target membrane receptor will be areceptor for biological signaling molecules, such as a GPCR receptor. Inthis case, the bound reagent ligand will often be the natural target, anagonist, or a synthetic analog of the biological signaling moleculesthat are bound by the target membrane receptor.

[0039] Alternative binding reactions are also possible, however. Forexample, the liposome bound target membrane receptor may be an enzyme.In this case, the reagent ligand may be a substrate or inhibitor of theenzyme. Alternatively, the liposome bound target membrane receptor mayitself be an enzyme substrate. In this case, the reagent ligand may bean enzyme, or a molecule with similar binding properties as the naturalenzyme that reacts with the target membrane receptor.

[0040] As yet another alternative, the liposome bound target membranereceptor may be an antigen, and the reagent ligand may be an antibodythat binds to the antigen.

[0041] As a third alternative, the target membrane receptor may be atransport protein, such as an ABC drug transporter protein, and thereagent ligand may be a ligand or analog to a ligand that is normallytransported by the transport protein.

[0042] As previously discussed, the target membrane receptor willusually be labeled with a moiety that is capable of emitting adetectable signal when exposed to an outside energy source. The lipidmembrane in turn will be tethered to an energy-emitting surface, wherethe energy emitted by the surface changes sharply as a function ofdistance away from the surface. Also tethered to this surface (bydifferent means) will be a reagent ligand that binds to the targetmembrane receptor. Due to the fluid nature of the lipid membrane, theresulting target membrane receptor and surface-bound reagent ligand arefree to associate and dissociate. Yet both remain attached to theenergy-emitting surface.

[0043] The geometry of the membrane, and the energy-emitting surface,are chosen so that the detectable signal-emitting moiety on the targetmembrane receptor is exposed to differing levels of excitation energydepending upon the binding or non-binding of the target membranereceptor to the surface-bound reagent ligand.

[0044] A number of different geometric configurations and detectionschemes are feasible for these purposes. One particularly favoredembodiment is the use of lipid membranes arranged in a sphericalliposomal configuration. These spherical liposomes may be tethered to anoptical surface that exposes the liposomes to evanescent waveexcitation. Here, there will be a gradient in excitation energy betweenthe surface side of the liposome, and the distal side of the liposome.The distribution of target membrane receptors on the liposome surfacecan thus be assessed by the relative intensity of the signal generatedby the detectible signal emitting moieties that are bound to the targetmembrane receptors.

[0045] Other membrane configurations, e.g. lipid layers mounted on asecond surface that projects away from an energy emitting surface,and/or other excitation sources (e.g. electrochemiluminescence, chemicalgradients, electron transport, resonance energy transfer, etc.), wherethere is a sharp decrease or increase in energy transfer as a functionof distance from the surface, may also be used.

[0046] This reagent and method exhibits a number of distinct advantagesover the prior art. One distinct advantage is that all of the chemistryrequired to perform the desired analysis is present on the support as asingle homogeneous unit. Thus for each test, no additional chemistry orprocessing steps (such as washing or centrifugation) are required.Because the reagent contains all necessary detection means, thetest-ligands do not need to be artificially labeled in any way. Theassay chemistry is inherently reusable, and, by simply applying a washstep between assays, may be reused for multiple assays with differenttest-ligands. The reagent and method uses several orders of magnitudeless reagents than previous art. Since the reagents used in this type oftest are typically quite expensive, the economic savings areconsiderable.

[0047] This support may contain a small or large number of differenttarget membrane receptors arranged on different sections of the support.In the case where only a small number of different types of targetmembrane receptors are present, liposomes containing the target membranereceptors may be applied to the support by simple dipping, coating,spraying, or other means. Alternatively, large numbers of differenttarget membrane receptors may be used, and the support may be amicroarray or flow cell that has hundreds or thousands of differenttarget membrane receptors. In this situation, the liposomes may beapplied by common microarray sample spotting methods, such as slottedpens, jet printing, and the like. In other situations, the support maybe the inside of a capillary tube, or the surface of an optical fiber,in which case more specialized fabrication methods may be required.

[0048] For evanescent detection methods, the support containing theenergy-emitting surface will normally be made of a solid material, suchas glass or plastic, that has an index of refraction that issignificantly different from the liposome containing aqueous media.However use of non-solid supports is also possible. As an example, highindex of refraction, water and oxygen permeable, porous polymers, suchas the materials used for soft contact lenses, may also be suitable.

[0049] In some cases, use of composite supports may be desirable. Suchcomposite supports may be composed of a first solid glass or plasticenergy emitting support layer that has an index of refraction that issignificantly different from the assay's aqueous media, covered with athin coating of a second material, such as a water permeable porouspolymer, which may have an index of refraction substantially similar tothe aqueous media. In this case, the underlying glass or plastic supportlayer may emit evanescent energy, while other test reagents in turn maybe coupled to a second layer coated on top of the energy emitting firstlayer. Such multilayer composites may be used to generate non-flatsurfaces where a single lipid bilayer may exist at a variable distanceaway from an energy-emitting surface.

[0050] The liposome reagents will typically need to be anchored to thesurface by means that are unaffected by the presence or absence oftest-ligands. Additionally, the target membrane receptors on theliposome will themselves usually be tethered to the surface byreversible linkages to surface bound reagent ligands.

[0051] Although in some cases, it may be preferable, for each differentliposome preparation, to perform this set of linkages by separate setchemical coupling reactions, this approach quickly becomes impracticalfor systems with a large number of different target membrane receptors.As a result, it is often preferable to employ an “active” supportsurface with a number of the “generic” components of the tetheredreceptor-ligand reagent (for example some of the tethering and/orbinding components) pre-prepared on the support surface. With an activesupport, a user may create the final microarray by simply spotting thevarious liposome preparations onto the active surface. The spottedliposome reagents can then form the appropriate anchoring and reversiblereagent ligand linkages with minimal additional effort.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 shows a diagram of a general-purpose microarray activesurface, and its interactions with a liposome reagent.

[0053]FIG. 2 shows a liposome in which the target membrane receptors,and a different set of anchor groups, are tethered to a surface, andobserved by evanescent wave techniques.

[0054]FIG. 3 shows a liposome in which the anchor groups remain tetheredto the evanescent wave-emitting surface, but the distribution of thetarget membrane receptors has been perturbed by an external agent (suchas a test-ligand).

[0055]FIG. 4 shows a liposome in which the target membrane receptors andanchor groups are tethered to a surface, and are observed by normalfluorescence illumination.

[0056]FIG. 5 shows a liposome in which distribution of target membranereceptors has perturbed by an external agent, again observed by normalfluorescence illumination.

[0057]FIG. 6 shows a combination normal fluorescence(epifluorescent)/evanescent illumination instrument used to read themembrane receptor microarray

[0058]FIG. 7 shows how a target membrane receptor can be tethered to anenergy-emitting surface by use of a bridging reagent ligand.

[0059]FIG. 8 shows how the coupling between target membrane receptors,and reagent ligands on an energy-emitting surface, can be disrupted byexcess amounts of test ligands.

[0060]FIG. 9 shows a multiple element membrane receptor microarray

[0061]FIG. 10 shows a flow cell incorporating multiple types of targetmembrane receptors.

[0062]FIG. 11 shows an example of a composite support material.

DETAILED DESCRIPTION OF THE INVENTION

[0063] Detection Methods:

[0064] Due to the relative scale of the distances involved, detectionschemes that are sensitive to positional changes over a liposomaldiameter range (50-5000 nm) are needed. For this purpose, techniquessuch as evanescent wave optics, electrochemiluminescence, electrontransport, surface plasmon resonance, and other short-range positionaldetection modalities are appropriate. Here, evanescent wave opticaldetection techniques will be explored in more detail.

[0065] As previously discussed, evanescent wave detection schemesfunction over several hundred nanometers. For the purposes of thisinvention, the essential factor is that the membrane component underinvestigation must be free to laterally diffuse in a direction roughlyperpendicular (towards or away) from an evanescent wave source, or othersharp gradient energy source.

[0066] One good way to achieve this end is to incorporate the membranecomponent into a lipid vesicle such as a liposome, and link the liposometo a surface that emits evanescent waves. Membrane components on theside of the liposome that is closer to the evanescent wave-emittingsurface will receive more excitation energy, while membrane componentson the side of the liposome that is further away from the evanescentwave-emitting surface will receive less excitation. Thus the relativeposition of the membrane components may be detected, yet the membranecomponents remain tethered to the liposome, and the evanescent waveemitting surface, throughout the assay.

[0067] Synthetic Liposome and Membrane Synthesis

[0068] Synthetic liposomes (or phospholipid vesicles) are usuallycreated by dialysis of the detergent dissolved target membranereceptors, and appropriate lipids, versus a non-detergent containingaqueous solution, using methods such as those of Bach, et. al.(Biochemistry 1986 Jul. 15;25(14):4007-20). This can lead to thespontaneous formation of liposomes with the target membrane receptorsembedded into the liposome's lipid bilayer membrane.

[0069] The hydrocarbon chain length of the lipids should be long enoughto enable a stable membrane structure to form in aqueous solutions, andshort enough so that lipids and integral membrane proteins componentsremain substantially mobile for at least one of the assay temperatures.Often these are lipid mixtures that may include phosphatidylserine,phosphatidylcholine, and/or phosphatidylethanolime. A number of theseconsiderations have been previously taught by Tans et. al., (Eur J.Biochem. 95: 449-457, 1979).

[0070] In order to obtain useful information, the distribution of thetarget membrane receptors must be detected by some means. To do this,the target membrane receptors are labeled with a reporter group, whichwill typically be a fluorescent or luminescent group. In the case of atransmembrane protein, for example, this reporter group may be acarboxy-terminal bound moiety. Alternatively, the target membranereceptors may be genetically engineered to additionally contain afluorescent or luminescent region, such as a green fluorescent proteinor aquelorin region. As a third alternative, a protein or factor thatnormally binds to the target membrane receptors may itself befluorescently or luminescently labeled, and the target membranereceptors detected indirectly. This protein or factor does not need tobe irreversibly bound to the target membrane receptor for all targetmembrane receptor conformational states. In some cases, it may bedesirable to use a label protein or factor that binds to the targetmembrane receptor preferentially when the receptor is in one type ofconformational state, but not in a different type of conformationalstate.

[0071] For GPCR target membrane receptors, this can be done byincorporating a second fluorescently labeled GPCR binding protein, suchas β-arrestin into the liposome. (Labeled β-arrestin is available fromNorak Sciences Corporation, Morrisville, N.C.). This can be done byincorporating the GPCR binding protein into the lipid dialysis mix atthe time of original liposome synthesis, followed by subsequent washingsteps to remove any leftover material adhering to the outside of theliposome.

[0072] To irreversibly affix the liposomes to an energy-emittingsurface, a separate set of anchor groups is usually required. A numberof methods may be used here, such as biotin-avidin or biotin-strepavidinbinding techniques. One way to do this is by biotin labeling a membranecomponent, and using it to “anchor” the membrane component to an avidingroup coupled to the surface. This anchor molecule may be a membranelipid, or an alternate membrane protein that is tightly bound to themembrane, but otherwise does not interact with the liposome's targetmembrane receptors. This liposome “anchor” molecule simply functions tohold the liposome in position, and is otherwise not active in the assay.Ideally, the liposome anchors will not otherwise interact with thetarget membrane receptors on the liposomes, nor with the test ligands,or reagent test ligands.

[0073] In this context, “irreversibly” and “irreversible” does not meanthat the liposomes or lipid membranes can never be removed from theenergy-emitting surface by any modality, but rather means that theliposomes or lipid membranes are expected to remain attached to theenergy-emitting surface for the duration of the assay.

[0074] Usually the anchor molecules will be biotinated or otherwisemodified before the liposome synthesis reaction, and then mixed with theappropriate liposome lipids, target membrane receptors, fluorescentlabeled target membrane receptor binding proteins (if any), anddetergents, and then dialyzed to form the completed liposome reagent.

[0075] Typically, a different liposome synthesis reaction will berequired for each different target membrane receptor.

[0076] Surface Binding Methods

[0077] In general, liposomes will be bound to the energy-emittingsurface by high affinity non-covalent bonds mediated by an intermediateset of linker-receptor groups. For example, as previously discussed, onepossible type of intermediate linker-receptor group is a biotin-avidinlinker. Alternative linker-receptor groups, such as antigen-antibodylinkers, will also frequently be used. Usually, two different methodswill be employed, one method used to simply anchor the liposome to thesolid surface in an irreversible manner, and the other method used tobind the reagent ligands to the surface. These reagent ligands, in turn,can reversibly bind to the target membrane receptors.

[0078] In order to keep the liposome membrane far enough away from theenergy-emitting surface, and avoid possible membrane rupture or lysis,it will often be preferable to place the anchor groups and reagentligand groups on the end of extended hyrophilic tethers. This allows theliposome to be bound to the solid surface, but at a far enough distanceto avoid lysis. An additional advantage of extended tethers is thisallows the underside of the liposome to be freely accessible to externalsoluble test ligands, which is required for various drug andligand-binding assays.

[0079] For tethering, methods such as thepolyethyleneglycol-phospholipid conjugate methods of Wagner and Taum(Biophysical journal 79, 2000, pp 1400-1414) may be used. Othertethering methods include the methods of Adimoolim et. al., J. Biol.Chem. 273(49), 1998, 32561-32567, MacBeath and Schreiber, Science 2000,289, 1760-1763; and Falsey, Renil et al. Bioconjugate Chem. 2001, 12,346-353. Generally, any hydrophilic tethering means that has aneffective length of between about 10 and 500 nm, and has appropriatesurface binding and receptor binding groups, will suffice.

[0080] Note that the entire tether does not necessarily need to behydrophilic. Rather the combination of the reagent-ligand and the tethermust simply be hydrophilic enough so as to enable the tetheredreagent-ligand to interact with the target membrane receptor in a waythat closely mimics the binding of the untethered reagent ligand to thetarget membrane receptor. Thus a strongly hydrophilic reagent ligand canbe used with a neutral or even a mildly hydrophobic tether. Similarlythe reagent ligand does not necessarily need to be at the end of thetether. Rather, multiple reagent ligands can be placed at variousdistances along the tether, much as a single fishing line can containmultiple hooks. Alternatively the reagent ligand may be placed at someintermediate point on the length of the tether, rather than at the end.

[0081] To reduce the work involved in creating microarrays with a largenumber of different target membrane receptors, it is helpful to put thegeneric parts of the reagent onto the energy-emitting surface. Here,such a pre-prepared surface is called an “active” surface. An example ofsuch a pre-prepared active surface is shown in FIG. 1.

[0082]FIG. 1 shows the details of a general-purpose “active” energyemitting surface constructed using general-purpose tethered reagents.Here an anchor receptor (1), such as avidin or strepavidin, that iscapable of binding to the liposome's anchor groups, is bound to surface(5) by tether group (2). The surface also contains means to presentreagent ligands to the liposome's target membrane receptors. Thesereagent ligands are presented so that the reagent ligands, while alwaysbound to the energy-emitting surface, are capable of binding to theliposome's target membrane receptor in a reversible manner.

[0083] Typically, the means to present reagent ligands will be a reagentligand receptor, such as an antibody (3), that in turn is bound toenergy emitting surface (5) by tether group (4). This reagent ligandreceptor is usually designed to bind to a reagent ligand with abifunctional “bridge” structure. Here, one part of the bridge reagentligand contains a haptein or epitope that binds to a receptor (3) (suchas an antibody) on the surface (5), and the second part of the bridgecontains the reagent ligand that binds to a liposome's target membranereceptor. Details of this “bridge reagent ligand” are shown in a laterfigure.

[0084] The anchor and reagent ligand receptors (1) and (3) willtypically be intermixed together on the same locations on energyemitting surface (5), so that a single liposome will encountersignificant amounts of each. The anchor and reagent ligand receptorswill be tethered to the surface with tether groups (2) and (4), that areusually long enough (typically around 10-500 nm) so that the liposome isable to float a safe distance away from the surface. This gap also(during the subsequent test) allows test-ligands to easily permeate tothe underside of the liposome.

[0085] The binding between the components on the active surface, and thecomponents on the liposome reagent, are shown in 11-20. Here, an anchorreceptor (11), such as avidin, tethered to the surface (15) by tether(12) binds to a liposome membrane anchor (21). This holds liposome (20)close to the energy-emitting surface. A reagent ligand receptor (13),such as an antibody, is tethered to the surface (15) by tether (14).This reagent ligand receptor then binds to the liposomes' targetmembrane receptor (22), usually through an intermediate bridge reagentligand (not shown). Because the scale of the liposome is considerablylarger than the scale of the other components, the liposome is drawn inan unnatural squashed configuration.

[0086] Microarray Preparation

[0087] If microarrays with large numbers of different target membranereceptors are desired, often it will be advantageous to prepare anactive surface, similar to that shown in FIG. 1, in advance.

[0088] To prepare the microarray, the various liposome solutions areexposed to appropriate bridge reagent ligands, which bind to the targetmembrane receptors. The unbound bridge reagent ligands are then removedby washing or dialysis. The liposomes are then spotted onto the activesurface by standard microarray spotting techniques, and the liposomesallowed to bind to the active surface via the anchor mechanisms. Thetarget membrane receptors are usually also reversibly linked to thesurface by way of the bridge reagent ligand link. If immediate use isnot desired, the microarrays may be stored for later use by furthertreatment with appropriate fixative solutions, such as trehalose,glycerol, or other preservative solutions.

[0089]FIG. 2 shows the details of a liposome bound to the surface of anevanescent wave-emitting surface. Here, the liposome (1) is anchored tothe surface (2) by the interaction between an anchor protein,carbohydrate or lipid embedded into the liposome lipid bilayer (3).Surface bound anchor receptors then bind this component (4).

[0090] Note that although in this example, the liposomes are bound by ananchor set of receptor-ligand interactions; other surface bindingmethods may also be used. As one alternative, the liposomes may bephysically entrapped next to the surface by a polymeric meshwork thathas small enough pores to entrap the very large liposome, but largeenough pores to enable test ligands to pass freely.

[0091] The liposome additionally contains a target membrane receptor(5), labeled with a detectable moiety (6), such as a fluorescent moiety.The energy-emitting surface contains a bound reagent ligand (7) capableof reversibly binding to the target membrane receptor (5).

[0092] In this example, the binding or non-binding of the targetmembrane receptor (5) to the reagent ligand (7) is detected byevanescent illumination (8). Here, the evanescent wave is strongest nearsurface (2), and rapidly decays as distance from surface (2) increases,so that there is an appreciable difference in illumination intensitybetween the top and bottom of vesicle (1), as is shown by arrow (9).Thus when the target membrane receptor (5) is bound to the reagentligand (7), its detectable moiety (6) is exposed to a high intensity ofevanescent illumination, and thus generates a strong fluorescent,luminescent, or other signal (10), which may be detected.

[0093]FIG. 3 shows how alterations in the binding between the targetmembrane receptor (5) and the reagent ligand (7) may be detected. Inthis example, a test ligand (11), which acts to disrupt the associationbetween the target membrane receptor (5), and the reagent ligand (7), isadded to the system. (Here, this disruption proceeds by test ligand (11)binding to target membrane receptor (5), and thus blocking reagentligand (7) from binding to target membrane receptor (5)). Thisdisruption breaks the reversible bond between the target membranereceptor (5) and its reagent ligand (7), and as a result, the targetmembrane receptor (5) is now free to diffuse away from theenergy-emitting surface (2), due to the fluid nature of the liposomelipid bilayer membrane (1). As a result, the detectable moiety (6) isnow, on the average, a greater distance away from the energy-emittingsurface (2), and is thus exposed to a significantly less amount ofevanescent illumination (8, 9). As a result, the fluorescent,luminescent, or other signal generated by moiety (6) is nowsignificantly decreased.

[0094] Regardless of the binding or non-binding of the target membranereceptor (5) to its reagent ligand (7), liposome (1) remains anchored tosurface (2) by the interaction between the anchor embedded into theliposome lipid bilayer (3), and anchor receptors bound to the surface(4).

[0095] Note that for simplicity, FIGS. 2 and 3 show the case where thetarget membrane receptor (5) is initially bound to surface (2) byreagent ligand (7), and where the test ligand (11) acts to disrupt thisbinding. The opposite situation, where the target membrane receptor (5)is not initially bound to surface (2) by reagent ligand (7), and wherethe test ligand (11) acts to promote the binding the target membranereceptor (5) to reagent ligand (7), is also quite feasible.

[0096] Test ligand (11) can alter the binding between the targetmembrane receptor (5) and the reagent ligand (7) by many differentmeans. Test ligand (11) may simply bind to target membrane receptor (5)directly and act to sterically hinder the binding between the receptor(5) and its reagent ligand (7), as is depicted in FIG. 2. Alternatively,test ligand (11) can alter binding between the target membrane receptor(5) and ligand (7) by other mechanisms, including binding to the reagentligand (7) itself, or by altering the conformation or state of thetarget membrane receptor (5) or the reagent ligand (7). Thesealterations of conformation or state can be mediated by enzymaticmodification, binding to allosteric receptor sites, etc.

[0097] Reference (Normalization) Signal Generation:

[0098] Because there are typically an uneven number of liposomesdeposited in any given microarray zone, each with differing numbers oftarget membrane receptors, and each with detector moieties of varyingefficiency, means to normalize the detectible signal to correct for allthese variables are highly important.

[0099] To do this, a normalization signal must be generated that is notaffected by the binding or non-binding of the target membrane receptorsto the reagent ligands, but otherwise is a function of liposome number,target membrane receptor number, detector moiety efficiency, etc. Theevanescent signal, which contains the target membrane receptor positioninformation, as well as all the other variables, can then be adjusted bythis normalization signal. The two signals can then be processed toreport the target membrane receptor position information, free fromdistortion by the other test variables.

[0100]FIGS. 4 and 5 show how normalization signals may be generated tohelp separate the evanescent signal depicted in FIGS. 2 and 3 fromextraneous background signals. FIG. 4 shows the case where the targetmembrane receptor remains bound to the reagent ligand, and FIG. 5 showsthe case where the binding between the receptor and ligand is disruptedby test ligands.

[0101] Here, for both FIGS. 4 and 5, a normalization signal is generatedby exposing the liposomes (1) to a control source of fluorescenceexcitation energy that does not vary as a function of distance fromsurface (2). For this purpose, a normal fluorescence excitation energysource, such as epifluorescence illumination from a microscope objectiveabove surface (2) may be used. This is shown by arrow (8). As a result,the energy (9) emitted by moiety (6) is relatively unaffected by thebinding or non-binding of the target membrane receptor (5) to reagentligand (7). This control or reference signal may be used to normalize,or otherwise process the signal depicted in FIGS. 2 and 3 to improve thesensitivity of the assay.

[0102] Non-evanescent control fluorescence excitation (8) may begenerated by a variety of means, including epiilluminescence excitationfrom above surface (2), or fluorescence illumination from below surface(2) at an angle other than the critical angles where evanescence effectsdominate.

[0103] Note that for FIGS. 4 and 5, as shown previously, liposome (1) isanchored to surface (2) by anchor (3) and anchor receptor (4). The testligand that disrupts the binding between target membrane receptor (5)and reagent ligand (7) is shown in FIG. 5 as (10).

[0104]FIG. 6 shows a detail of the experimental apparatus used to obtaindata from the membrane microarrays shown in FIGS. 2-5. Here the membranemicroarray (1) is first illuminated by a fluorescent microscope (2) inorder to obtain a normalization (reference) signal. This fluorescentmicroscope contains a fluorescent light source (3), which sendsillumination through a bandpass mirror (4). This illumination is focusedby objective (5) onto the microarray. The return fluorescence signalpasses back through objective (5) though bandpass filter (6) and into adetector, such as a digital camera (7).

[0105] The evanescent illumination is provided by a second evanescentlight source (10). Here, a second illuminator, such as a 488 nm argonlaser (11), sends illumination through a collimator (12), which isdirected by a mirror (13) to illuminate the underside of the microarray(1) with illumination (14) at the correct angle for evanescentillumination. The liposomes on the microarray (1) then emit anevanescent stimulated fluorescent signal which is imaged by the sameoptical system (5), (4), (6), (7) used to detect the normalization(reference) signal. Note that in operation, the reference fluorescentsignal (used for normalization) and the evanescent fluorescent signalare obtained sequentially, rather than simultaneously.

[0106]FIG. 7 shows the details of how a bridging reagent ligand may beused to bind a target membrane receptor to a microarray surface. Hereliposome (1) has its target membrane receptor (2) bound to microarraysurface (3) by a bridging reagent ligand (4) that binds both themembrane receptor (2), and a microarray surface bound receptor (5) (suchas an antibody), tethered to microarray surface (3) by tether (6).Usually target membrane receptor (2) is labeled with a fluorescentmoiety or reporter group (7).

[0107] The bridging reagent ligand (4) will often contain two groups.One group (10) is a reagent ligand that binds to target membranereceptor (2). A second group (11) contains means to link the bridgingreagent ligand (4) to the microarray surface. In this example, thesecond group (11) contains a ligand (haptein, epitope, etc.) that bindsto the microarray surface bound receptor (5). Here, this receptor (5) isan antibody against the bridge reagent ligand's haptein or epitopeportion. Liposome (1) is additionally bound to microarray surface (3) byanchor means (20).

[0108]FIG. 8 shows how the binding between a target membrane receptor ona liposome, and a microarray surface receptor, normally mediated by abridging reagent ligand, may be disrupted by the addition of excessamounts of a target ligand. Here liposome (1), previously had its targetmembrane receptor (2) bound to microarray surface (3) by a bridgingreagent ligand (4) bound to a microarray surface bound receptor (5)(such as an antibody), tethered to microarray surface (3) by tether (6).As before, target membrane receptor (2) is labeled with a fluorescentmoiety or reporter group (7). Also as before, liposome (1) is bound tomicroarray surface (3) by an anchor means (20).

[0109] Here, however, excess amounts of a test ligand (30) are added tothe microarray. Test ligand (30) binds to target membrane receptor (2),as shown by (31). This disrupts the binding between target membranereceptor (2) and the bridging reagent ligand (4). As a result, targetmembrane receptor (2) is free to diffuse away from the microarraysurface (3). As a result, fluorescent moiety or reporter group (7) alsodiffuses further away from the microarray surface (3), and thus receivescorrespondingly less evanescent illumination. Thus, if test ligand (30)binds to target membrane receptor (2), the evanescent excitedfluorescent signal emitted by (7) will decrease in response to anincreasing concentration of test ligand (30).

[0110]FIG. 9 shows a perspective view of a multi-element membranereceptor microarray. Here samples of different liposome preparations arespotted onto a microarray surface (1). Each liposome preparation isitself homogeneous. However different preparations will differ invariables such as the target membrane receptor type, type of bridgingreagent ligand, lipid or cofactor composition, etc. For example, ahypothetical serotonin receptor microarray might contain one liposomepopulation of 5-HT_(1A) receptors (2), a second liposome population of5-HT_(2B) receptors (3), a third liposome population of 5-HT_(2C)receptors (4), a fourth population of 5-HT₆ receptors (5), and so on. Itcould additionally contain either a homogeneous population of 5-HTbridging reagent ligands, such as 5-HT-haptein groups, or alternativelycontain a population of different 5-HT bridging reagent ligands,comprised of different 5-HT analogs. This would allow discriminationbetween different test ligands with differing 5-HT receptor affinities.

[0111] A test ligand (6) is applied to the microarray, and differencesin the evanescent illumination excited fluorescent signal are observed.Appropriate reference normalization signal data are also obtained.Often, microarray (1) will be a component of a flow-cell, so thatdifferent test ligand samples and controls may be applied to themicroarray during the course of an experiment.

[0112]FIG. 9 also shows a single isolated liposome (7) as a scalereference.

[0113]FIG. 10 shows a reusable flow cell containing a membrane liposomemicroarray (1), and a transparent cover (2). Samples, such as variousdrug discovery candidate test ligands, wash buffers, control samples,etc. flow through the cell on a sequential basis (3). Here, the flowcell contains both a drug screening section (4) containing samples ofdifferent target membrane receptors on different liposomes, and an ADMETsection (5) that gives immediate feedback as to the potentialsuitability of a given candidate test ligand for drug use. Here, theADMET section contains a portion with various cytochrome P450 drugdetoxification membrane proteins (such as CYP3A4) on different liposomes(6), and a portion with various ABC drug transporter membrane proteins(such as P-glycoprotein) (7). Other ADMET detectors may also be added asappropriate.

EXAMPLES Example 1 Evanescent Wave Calculations

[0114] Upon hitting a boundary between two media with different indexesof refraction, there is a critical angle in which light waves can eitherpass from the first media to the second media, or bounce back into thefirst media. At angles where light bounces back, part of the wave energypasses into the second media for a very short distance. This is calledthe evanescent wave. Evanescent wave techniques are commonly used inmicroscopy to help visualize regions where cultured cell membranesadhere to transparent supports.

[0115] In the common situation where light is passing from a glass ortransparent support into an aqueous media, evanescent waves typicallypenetrate several hundred nanometers into the aqueous media. The wavedecays in intensity at a rate of

Intensity=I₀e^((−z/d))

[0116] Where I₀ is the illumination intensity at the support surface, zis the distance in nanometers above the support surface, and d is aconstant (approximately 277 nanometers). (For these calculations, “e”will be approximated as 2.718.)

[0117] Thus for a roughly spherical 2 micron (2000 nm) diameter “large”liposome anchored to a microarray surface, the evanescent illuminationintensity across the various portions of the liposome will be roughly asshown in Table 1 below: TABLE 1 Evanescent illumination intensity acrossa 2 micron diameter liposome Average Evanescent Liposomal quadrantdistance intensity Microarray bound base   0 nm  100% Lower quarter(microarray side)  250 nm 40.56%  Lower-middle quarter  750 nm 6.67%Upper-middle quarter 1250 nm 1.10% Upper quarter 1750 nm 0.18%

[0118] If all of the liposomes' fluorescently labeled target membranereceptors are bound to the surface by reagent ligands, then the liposomewill fluoresce with a normalized intensity of 100%

[0119] By contrast, if the liposome's fluorescently labeled targetmembrane receptors are displaced from the surface by test ligands, thereceptors are then free to randomly diffuse throughout the microarraysurface. In this situation, the fluorescent intensity (roughly adjustedfor variations in liposome surface area as a function of the quadrantsize) is approximately as shown in table 2 below: TABLE 2 fluorescenceof membrane receptors uniformly distributed on an evanescentlyilluminated 2 micron liposome Illumination Apx. % surface Liposomalquadrant intensity area Total Lower quarter 40.56% 12.5% 5.07%Lower-middle quarter 6.67% 37.5% 2.50% Upper-middle quarter 1.10% 37.5%0.41% Upper quarter 0.18% 12.5% 0.02% Total intensity 8.01%

[0120] Thus for a 2 nm liposome, the fluorescence of the randomlydiffusing target membrane receptors, displaced from the surface by thetest ligands, will decrease to about 8% of their initial surface-boundvalue. Larger diameter liposomes will produce correspondingly largereffects. For example, target membrane receptors distributed randomlyover a 5-micron diameter liposome will decrease in intensity to onlyabout 1.35% of their original surface-bound value.

[0121] Synthetic liposomes typically incorporate membrane proteins with50% of the membrane receptors oriented on the “right side” (“N” terminaloutside of the liposome), and 50% of the receptors on the “wrong side(“C” terminal outside of the liposome). This effect diminishes thesignal only slightly, however. This is because the incorrectly insertedreceptors randomly distribute throughout the liposome surface. Thus, forexample, for 2 nm liposomes where both the “right side” and “wrong side”receptors are equally fluorescently labeled, the signal-to-noise ratiochanges from 1/0.08 to (0.5+0.04)/0.08. This represents a decrease inthe (bound/unbound) signal from a “good” ratio of 12.5:1, to a “stilldecent” 6.75:1.

Example 2 Signal Processing

[0122] As previously discussed, one of the most important signalenhancing techniques is signal normalization, using concurrent normalfluorescence (epifluorescent) illumination to provide a secondreference, signal. Here the microarray spots are sequentially visualizedby both evanescent illumination and epifluorescence illumination, andthe evanescent signal is normalized by the epifluorescent signal. Sincethe epifluorescent signal is relatively unaffected by the position ofthe membrane proteins on the liposome, epifluorescence normalizationcorrects for many variables, including liposome spot density, labelingefficiency, and GPCR receptor concentration.

[0123] As an example of the issues that are involved in signalprocessing, consider the following model:

[0124] Any given microarray spot will have a variable number ofliposomes “x”, and a background signal “b”, where b may be due tocontaminating lysed lyposomes, autofluorescence, or other impurities inthe reagent.

[0125] Each liposome, in turn, will generate an evanescent fluorescentsignal, E, that is proportional to the relative ratio of the liposome'sfreely diffusing target membrane receptors (R_(f)) to the surface boundmembrane receptors (R_(b)).

[0126] Thus E=m(R_(b)/(R_(f)+R_(b))) where m is an efficiency constant.

[0127] In fractional terms: R_(f)+R_(b)=1 so therefore on a per liposomebasis:

E=m(1−R _(f)).

[0128] So the total signal “S” from any given microarray spot will beproportional to the output per liposome, “E” times the number ofliposomes, “x” so that:

S=m(1−R _(f))x+b

[0129] To determine the percentage of free receptors, R_(f) for anygiven microarray spot, the efficiency constant “m”, the number ofliposomes “x”, and the background signal “b” must first be determined.

[0130] Here, the normal fluorescence (normalization) signal, “N” isuseful, because this signal is unaffected by the ratio of bound to freetarget membrane receptors. Assuming that the normalization signal isdone at the same wavelength, and that the instrument is previouslycalibrated to correct for any differing efficiencies between the normalfluorescence and evanescent signals, then:

N=m(R _(f) +R _(b))x+b and since R _(f) +R _(b)=1; then

N=mx+b

[0131] Subtracting the evanescent signal from the normal fluorescentsignal thus gives a result proportional to the number of free receptors,R_(f), because:

N−S=mx+b−[m(1−R _(f))x+b]

Thus: N−S=m(R _(f))x

[0132] If the background signal, “b” is small, the fraction of unboundtarget membrane receptors R_(f) per microarray spot can be found bydividing this result by the normal fluorescent signal, N, becauseaccording normal mathematical approximations, for any “b” that is small:

1/(1+b)≅1−b

Therefore: m(R _(f))x/(mx+b)≅R _(f) −bm(R _(f))x

[0133] so for small values of b, where bm(R_(f))x is small, then:

R _(f)≅(N−S)/N  (1)

[0134] or alternatively

R _(b)≅1−(N−S)/N  (2)

[0135] In the case where the background signal “b” is large, or whereadditional precision is required, other signal processing methods may beused. One good way to do this is by incorporating the reagent spots intoa flow cell. Here, each reagent spot may be calibrated by first exposingthe reagent to a low-level control solution containing no test ligands,and determining a first control evanescent signal S₁. The flow cell isthen exposed to a high level control solution containing a saturatingconcentration of test ligands, and a second control evanescent signal S₂is obtained. Since for the zero test ligand control case, S₁, all thetethered membrane receptors are bound to the reagent ligands; soR_(f)=0, and R_(b)=1. Thus:

S ₁ =m(1−0)x+b; or alternatively S ₁ =mx+b

[0136] By contrast, for the high test ligand control case S₂, all thetethered membrane receptors are free to diffuse away from the reagentligands, so R_(f)=1 and R_(b)=0. Thus:

S ₂ =m(1−1)+b; or alternatively S ₂ =b

Thus S ₁ −S ₂ =mx;

[0137] To determine the percentage of free (R_(f)) or bound (R_(b))tethered membrane receptors for intermediate levels of unknown testligands producing an evanescent signal S_(test) the S_(test) signal isprocessed using the data obtained from the S1 and S2 control data by

S _(test) =m(1−R _(f))x+b

and since S ₁ −S ₂ =mx; and S ₂ =b, then

S _(test)=(S ₁ −S ₂)(1−R _(f))+S ₂

[0138] Solving for the percentage of free receptors, R_(f), for anygiven value of S_(test) gives:

(S _(test) −S ₂)/(S ₁ −S ₂)=(1−R _(f))

[0139] giving

R _(f)=1−(S _(test) −S ₂)/(S ₁ −S ₂)  (3)

[0140] After the flow cell is calibrated with the low and high controlsolutions, and the S₁ and S₂ values are recorded for each reagent spot,the flow cell is then regenerated by flushing out the high control testligands with excess buffer. After the regeneration cycle, theexperimental test ligands are then added to the system, and the“S_(test)” experimental signal levels processed using the previouslyobtained S₁ and S₂ control values as in equation (3) above.

[0141] More complex processing schemes, involving combinations of normalflurorescence data, as well as high and low control flow cell data, arealso possible.

Example 3 Studies with Model GPCR Target Membrane Receptors

[0142] Not all GPCR receptors bind drug ligands. Some, such asbacteriorhodopsin, act as sensors. Bacteriorhodopsin is a7-transmembrane protein with well-understood properties. It is availablein low cost and large quantities from a variety of commercial sources.It is easy to work with, and is often used for exploratory biophysicalresearch. Here, methods to construct prototype membrane sensors usingliposomes with fluorescent bacteriorhodopsin are described.

Preparation of Large Liposomes Containing Fluorescent Bacteriorhodopsin

[0143] Bacteriorhodopsin from commercial sources can be labeled with theAlexa Fluor 488 fluorophore (a high efficiency fluorescent moiety), andincorporated into giant (5 micron) phospholipid vesicles (liposomes)following the methods of Kahya et. al (Kahya N, Pecheur E, de Boeij, W,Wiersma D, Hoekstra D, “Reconstitution of Membrane Proteins into GiantUnilamellar Vesicles via Peptide-Induced Fusion”, Biophysical Journal,2001 81: 1464-1474). In order to anchor these liposomes to energyemitting surface, trace amounts of N-biotinyl phosphatidylethanolamine,following the methods of Adimoolan et. al., (Adimoolam S, Jin L, GrabbeE, Shieh J, Jonas A, “Structural and Functional Properties of TwoMutants of Lecithin-Cholesterol Acyltransferase (T123I and N228K)”,Journal of Biological Chemistry, 1998 272(49): 32561-32567), should beadded to the liposome synthesis mix. The N-biotinylphosphatidylethanolamine serves as a biotinated anchor group, which canbe used to anchor the liposomes to an energy-emitting surface.

Active Energy Emitting Surface Preparation

[0144] An active energy-emitting surface can be constructed usingmicroarray slides, following the methods of Macbeath (MacBeath G,Schreiber S “Printing proteins as microarrays for high-throughputfunction determination” Science, 2000, 289(5485):1760-3). Here, BovineSerum Albumin (BSA) is used as a tether to link receptors to themicroarray slide glass surface. This BSA linking method enables themicroarray bound receptors to move somewhat, and additionally helpsprevent direct contact between the liposome and the microarray surface.To do this, serum albumin is covalently bound to silane treated glassslides. This BSA layer, in turn, is activated with N,N′-disuccinimidylcarbonate. This activates the lysine, aspartate, and glutamate residuesof the BSA, which in turn, are then available to react with surfaceamines on other proteins.

[0145] After activation, the BSA layer on the microarray surface isfurther reacted with a solution containing both strepavidin and IgGantibody. The strepavidin and IgG form covalent urea (or amide) linkswith the BSA, and thus become covalently attached to the microarraysurface. After the reaction is complete, the slides are then rinsed withexcess glycine to quench any unreacted groups.

[0146] Typically, in these experiments, the IgG antibody used is eitherrabbit anti-mouse IgG, or a control rabbit anti-goat IgG.

[0147] Binding of liposomes to microarray plates: Liposome solutions aretypically stored in 40% glycerol following the methods of Macbeath, andspotted directly onto the microarray surface using a mechanical pinmicroarrayer device. The plates are incubated at 37° C. in a 95%humidity chamber for 1 hour, rinsed with phosphate buffered saline(PBS), and then stored in PBS at 4° C. until use. Often, it is useful toadditionally spot 1-micron diameter Alexa Fluor 488 labeled latexmicrospheres to strategic microarray locations asevanescence/fluorescence controls. The liposomes are anchored to themicroarray surfaces through the strepavidin—biotin link. By contrast,the fluorescently labeled bacteriorhodopsin target membrane receptors onthe liposomes will not initially bind to the microarray plate becausethe rabbit anti-mouse IgG does not directly bind to bacteriorhodopsin.

Instrumentation

[0148] To observe the membrane microarrays, a standard Leitzfluorescence microscope, with a lower stage modified to allow forevanescent illumination, may be used. The microarray is alternatelyilluminated with the evanescent light source (see FIG. 6), and thefluorescence light source, and digital micrographs may be taken (NikonCoolpix 995 in time exposure mode). The data is then downloaded andanalyzed.

Characterization Experiments

[0149] In one experiment, microarrays can be created in which thesurface contains a first type of zone where the microarray-bound IgG isa rabbit anti-mouse antibody, and a second type of zone where themicroarray bound IgG is a control rabbit anti-goat antibody. Alexa fluor488 labeled bacteriorhodopsin liposomes can be spotted on both types ofmicroarray zones, and allowed to anchor. The microarray will then beincubated with either a solution of monoclonal mouseanti-bacteriorhodopsin antibody or a control monoclonal mouse antibodyat 37° C. for one hour, and observed by combinationevanescence/fluorescence microscopy. Here, the only test condition wherebacteriorhodopsin will tightly bind to the microarray surface is thezone where the mouse anti-bacteriorhodopsin antibody is bound to therabbit anti-mouse antibody. The mouse anti-bacteriorhodopsin antibodybinds to bacteriorhodopsin, and the antibody labeled receptors move inthe fluid liposome membrane and become bound to the microarraysurface-bound rabbit anti-mouse antibody.

[0150] The digital signals obtained from the microarray spots areaveraged and normalized. The results can then be expressed according toequation (2): Bound bacteriorhodopsin

(R _(b))=1−[(Fluorescent signal−Evanescent signal)/Fluorescent signal]

[0151] This will normally produce results, such as shown in table 3below: TABLE 3 binding of bacteriorhodopsin to microarray antibody zonesAdded anti-bacteriorhodopsin antibody Mouse monoclonal anti- Controlmouse monoclonal Antibody bacteriorhodopsin: antibody: bound to % Boundbacteriorhodopsin % Bound bacteriorhodopsin microarray: R_(b) R_(b)Rabbit N = 100, E = 90, therefore N = 200, E = 20, therefore anti-mouseR_(b) = 1 − (100 − 90/100) R_(b) = 1 − (200 − 20)/200 antibody zoneR_(b) = 90% bound R_(b) = 10% bound Control N = 150, E = 15, therefore N= 50, E = 50, therefore rabbit antibody R_(b) = 1 − (150 − 15)/150 R_(b)= 1 − (50 − 5)/50 zone R_(b) = 10% bound R_(b) = 10% bound

[0152] Note that in the above example, the amount of material per spot,as shown by the normal fluorescent intensity “N”, is shown varying from50 to 200 units. However normalization methods can compensate for thesevariations.

[0153] Adding excess control monoclonal antibody to the microarray willtypically reverse the bacteriorhodopsin binding. In an alternate versionof this experiment, which simulates the use of bridging reagent ligandtechniques, fluorescent bacteriorhodopsin liposomes can first beincubated with monoclonal mouse anti-bacteriorhodopsin antibody, andthen bound to strepavidin+rabbit anti-mouse coated zones on microarrayactive surfaces. Here, the liposomes will anchor to the microarraysurface by the strepavidin-biotin links. The mouse monoclonal antibody,which itself is bound to the liposome's bacteriorhodopsin targetmembrane receptors, in turn binds to the rabbit anti-mouse antibody zoneon the microarray surface. The fluorescent bacteriorhodopsin targetmembrane receptors move in the fluid liposome membrane and become boundto the surface, resulting in a larger evanescent excited fluorescencesignal.

[0154] This binding can be broken by an excess of control mousemonoclonal antibody (e.g. without bacteriorhodopsin binding activity).The control antibody binds to the surface bound rabbit anti-mouseantibody, and saturates all of its anti-mouse binding sites. Thus themouse anti-bacteriorhodopsin antibody on the liposome can no longer bindto the surface, and the fluorescent bacteriorhodopsin molecules are nowfree to diffuse randomly throughout the entire liposome surface,resulting in a drop in the evanescent excited fluorescent signal. Here,the control antibody has a competitive effect similar to the competitiveeffect of the test ligands discussed previously. An example of this typeof competition experiment is shown in table 4 below: TABLE 4 effect ofcompetition binding Added control mouse antibody Antibody Excess mouseantibody No excess mouse antibody bound to % Bound bacteriorhodopsin %Bound bacteriorhodopsin microarray: R_(b) R_(b) Rabbit N = 50, E = 5,therefore N = 100, E = 90, therefore anti-mouse R_(b) = 1 − (50 − 5/50)R_(b) = 1 − (100 − 90)/100 antibody R_(b) = 10% bound R_(b) = 90% boundControl N = 200, E = 20, therefore N = 150, E = 15, therefore rabbitantibody R_(b) = 1 − (200 − 20)/200 R_(b) = 1 − (150 − 15)/150 R_(b) =10% bound R_(b) = 10% bound

[0155] As before, normalization techniques may be used to compensate forthe effect of varying spot intensity.

[0156] Table 4 shows that excess control mouse monoclonal antibody candisplace the surface bound mouse anti-bacteriorhodopsin antibody. Thusthe fluorescent bacteriorhodopsin molecules on the liposome are free todiffuse away from the microarray surface, resulting in a smallerevanescent excited fluorescent signal.

Example 4 Construction of Membrane Receptor Microarrays for DrugDiscovery Purposes

[0157] A drug discovery membrane receptor microarray may be constructedusing commercially available cloned membrane receptors and bridgereagent ligands. Here, a prototype multi-element microarray containingvarious types of serotonin (5-HT) receptors is described.

[0158] Serotonin receptors play an important role in the nervous system,and are responsible for a variety of behavior disorders includingappetite, depression, and obesity. This receptor family is the subjectof much intense drug discovery effort by the pharmaceutical industry,and methods to facilitate such discovery methods are of large practicalinterest.

[0159] Serotonin membrane receptor microarrays can be constructed usingcommercially available serotonin GPCR receptors such as 5-HT_(1A),5-HT_(2B), 5-HT_(2C), 5-HT₆, and 5-HT₇. These are available from theEuroscreen corporation, and other companies. As a control, themicroarray may additionally contain a number of “non-target” receptorsfrom an entirely different GPCR receptor family, such as the dopaminereceptor family. These non-target receptors can serve as a control, andalso help detect unwanted target ligand cross reactions.

[0160] For these prototype microarrays, pure (no cofactor) GPCRreceptors may be used. However more physiologically realistic systemsmay be created by incorporating additional receptor cofactors (such asGα, β, and γ-proteins, etc.) into the liposome preparations asappropriate. Some of the considerations as to the physiological impactof such cofactors is discussed in Brys et. al., Reconstitution of theHuman 5-HT1D Receptor-G-Protein Coupling: Evidence for ConstitutiveActivity and Multiple Receptor Conformations, Molecular Pharmacology2000, MOL 57:1132-1141).

GPCR Fluorescence Labeling Methods

[0161] For these purposes, direct fluorophore labeling by chemical means(such as the Alexa Fluor 488 labeling method discussed for thebacteriorhodopsin examples), although often useful, may sometimes beless preferred in cases where such labeling damages the receptors. Useof GPCR-fluorescent protein fusion products, such as GPCR-greenfluorescent protein, or GPCR aquelorin products, although quitefeasible, is labor intensive because this requires the creation of adifferent fusion product for each microarray element. As a result, useof modified forms of natural wide-specificity GPCR binding proteins,such as fluorescent β-arrestin (Ferguson, S., Barak, L., Zhang, J.,Caron, M, “G-protein-coupled-receptor regulation: role ofG-protein-coupled-receptor kinases and arrestins” Can. J. Physiol.Pharmacol. 1996, 74: 1095-1110) may be useful. This is becauseβ-arrestin binds to the vast majority of the various GPCR proteins.

[0162] To generate a higher fluorescent or luminescent signal, use offluorescent micro particles may be advantageous. Such particles, such asquantum dots (quantum dot corporation, Hayward Calif.), fluorescentlatex micro spheres (transfluospheres, Molecular Probes Inc., EugeneOreg.), etc. typically have diameters between about 10 nm and 100 nm,contain large numbers of signal generating molecules, and can be labeledwith antibodies specific to the cytoplasmic side of various targetmembrane receptors, or target membrane receptor binding proteins. Use ofsuch micro particle based label moieties can produce a more intensesignal requiring less amplification and lower cost detection equipment.

[0163] Liposome synthesis: in this example, liposomes are synthesized bydialysis of detergent solubilized purified 5-HT receptors, liposomelipids, biotinated lipids (for microarray anchoring purposes), andfluorescent labeled wide-specificity GPCR binding proteins such asGFP-β-arrestin. This results in the creation if intact liposomescontaining labeled serotonin receptors and suitable anchor groups. Adifferent synthesis is done for each different serotonin receptor.

Bridge Reagent Ligands

[0164] In order to be useful for drug discovery purposes, the liposome'sfluorescent serotonin GPCR receptors must bind to the microarray surfacein the absence of competing test ligands, and dissociate from thesurface in the presence of competing test ligands. As previouslydiscussed, this can be done by using bridge reagent ligands. These arecomposed of a conjugate of a ligand that binds to the appropriateserotonin GPCR receptors, and a chemical group (such as a haptein) thatbinds to a haptein-receptor (usually an anti-haptein antibody) that istethered to the microarray surface.

[0165] Although many different types of haptein (or other tethering)groups may be used for the 5-HT-haptein conjugates, use of the coumaringroup is particularly convenient because a number of 5-HT-coumarinconjugates have been previously synthesized by other workers. Previoussynthesis of this type have been reported by Friedrich et. al.(“Investigation of the 5-HT3 Serotonin Receptor Using Novel FluorescentLigands”, Ecole Polytechnique Federale de Lausanne (poster session)),and a number are commercially available from Tocris Cookson Inc., andother vendors. Although the coumarin group is itself fluorescent, it isexcited by shorter wavelengths than those used by the GPCR labelfluorophores, and thus cross-talk effects are minimal. These coumaringroups can be used as hapteins because they are antigenic, andanti-coumarin antibodies are commercially available.

[0166] Here, the liposome preparations are exposed to the 5-HT-coumarinbridge reagent ligands, and the excess (unbound) bridge reagent ligandsare then removed by washing. The liposomes+bound bridge reagent ligandsare then spotted onto different locations on an active microarraysurface.

Active Microarray Surface Preparation

[0167] The microarray surface chemistry plays a critical role forevanescent-liposome microarrays. Here there are two generalconsiderations. The first is that both the microarray's anchor receptorsand reagent ligand receptors should preferably be attached to thesurface by tether groups that allow a sufficient aqueous gap between thefragile liposome and the surface so that the liposome does not lyse uponthe surface, and so that test-ligands can penetrate to the underside ofthe anchored liposome.

[0168] For these purposes, the polyethylene glycol tethers of Vermetteet. al. are useful (Vermette P, Meagher L, Gagnon E, Griesser H J,Doillon C J “Immobilized liposome layers for drug delivery applications:inhibition of angiogenesis” J Control Release 2002, 80(1-3):179-95).This work has shown that the use of polyethylene glycol-biotin linkersimproves the lifetime of intact liposomes on plastic surfaces. Otherhydrophilic tethering molecules may also be used, however.

[0169] For these experiments, appropriate active surfaces can be createdby preparing a microarray surface containing a mixture of avidin (orstrepavidin) groups bound to the surface by a polyethylene glycoltethers, and a mixture of anti-coumarin antibodies, also bound to thesurface by a polyethylene glycol tether.

[0170] To make a useful drug discovery microarray reagent, a combinationof liposome preparations containing different populations of 5-HTreceptors, control receptors, and different types of bridge reagent 5-HTligands is spotted onto a microarray surface, creating a prototypemicroarray reagent as shown in table 5 below: TABLE 5 prototypeserotonin receptor microarray Bridge Alternate 5-HT Reagent - BridgeReagent Alternate 5-HT Receptor Ligand Ligand 1 Reagent Ligand 25-HT_(1A) 5-HT-coumarin Low affinity analog High affinity analog5-HT_(2B) 5-HT-coumarin Low affinity analog High affinity analog5-HT_(2C) 5-HT-coumarin Low affinity analog High affinity analog 5-HT₆5-HT-coumarin Low affinity analog High affinity analog 5-HT₇5-HT-coumarin Low affinity analog High affinity analog D₁ Dopamine- Lowaffinity analog High affinity analog (dopamine) coumarin D₂ longDopamine- Low affinity analog High affinity analog (dopamine) coumarin

Prototype Drug Discovery Assays

[0171] The microarray shown in table 5 may then be incorporated into aminiature flow-cell, constructed using a microscope slide, appropriatespacers, tubing, and a cover slip so that different reagents may beslowly flowed over the microarray. The microarray, in turn, is mountedonto a dual evanescent/fluorescent detection apparatus, similar to thatshown in FIG. 6. The microarray may then be calibrated by exposing it tothe appropriate control solutions with zero and high levels of 5-HT, asdiscussed previously.

[0172] After the high control solution has been flushed out, and themicroarray target membrane receptors restored to their no-target-ligand,surface-bound configuration, samples of appropriate test ligands canthen be applied to the microarray, and the microarray responsecharacterized.

[0173] As an example, consider a test-ligand that is a 5-HT analog withhighest affinity for the 5-HT_(2C) receptor, and relatively low affinityfor the other 5-HT receptors. Its response in the membrane microarrayflow cell may look like table 6 below: TABLE 6 binding of a 5-HT_(2C)specific test ligand to a serotonin receptor microarray Alternate highAlternate low affinity 5-HT affinity 5-HT Receptor Bridge Reagent BridgeReagent Bridge-Reagent 5-HT_(1A) R_(b) = 90% R_(b) = 95% R_(b) = 70%5-HT_(2B) R_(b) = 90% R_(b) = 95% R_(b) = 70% 5-HT_(2C) R_(b) = 10%R_(b) = 20% R_(b) = 5% 5-HT₆ R_(b) = 90% R_(b) = 95% R_(b) = 70% 5-HT₇R_(b) = 90% R_(b) = 95% R_(b) = 70% D₁ (dopamine) R_(b) = 100% R_(b) =100% R_(b) = 100% D₂ long (dopamine) R_(b) = 100% R_(b) = 100% R_(b) =100%

[0174] In this example, the experimental target-ligand displaces the5-HT_(2C) target membrane receptors the most. The example also showsthat the target ligand also has mild cross reactivity with other membersof the 5-HT receptor family, but no cross-reactivity whatsoever withdopamine family target membrane receptors.

ADMET Assays Example 5 P-glycoprotein Transport Assay

[0175] Although much of the previous discussion, as well as theserotonin receptor example, have focused on assays in which thesurface-bound reagent-ligand binds to the target membrane receptor viaintermediate bridge reagent ligands, this need not always be the case.Sometimes the surface may contain a tethered “non-drug-like” reagentligand, such as an antibody, that directly binds to the target membranereceptor.

[0176] As an example of this alternative method, consider P-glycoproteinassays. P-glycoprotein is an ABC (ATP binding cassette) protein thatplays a major role in pumping drugs out of cells. Indeed,hyperexpression of P-glycoprotein is an important mechanism of cancercell resistance to anticancer drugs. In this role, it is often referredto as the “multidrug resistance protein 1”, or MRP1. Due to its majorrole in controlling drug transport in the body, characterization of anexperimental test ligand's ability to be transported by P-glycoproteinis an important ADMET test.

[0177] P-glycoprotein is a transmembrane protein that pumps drugs out ofthe cell, against a concentration gradient, by the hydrolysis of ATP.The transport process involves several steps in which the protein bindsthe drug, changes conformation to expel the drug, binds ATP, and thenregenerates it's original conformation. Previous workers, (Nagly et.al., P-glycoprotein conformational changes detected by antibodycompetition, Eur. J. Biochem. 268, 2416-2420 (2001)) have producedmonoclonal antibodies, such as UIC2, that bind to the conformation thatthe P-glycoprotein assumes the presence of P-glycoprotein transportsubstrates or inhibitors. The antibodies do not bind to the conformationthat P-glycoprotein assumes in the absence of such transport substratesor inhibitors.

[0178] Such conformational sensitive antibodies may be used as reagentligands to perform P-glycoprotein ligand transport assays in theevanescent liposome format. Here, as always for this format,P-glycoprotein needs to be labeled with a detectible moiety. To do this,a number of methods are suitable, such as the green fluorescent or cyanfluorescent protein labels. Here, the methods of Rajagopal et. al., (Invivo analysis of human multidrug resistance protein 1 (MRP1) activityusing transient expression of fluorescently tagged MRP1 Cancer Res 2002January 15;62(2):391-6), may be used.

[0179] Next, the fluorescently tagged P-glycoprotein molecules arereconstituted into artificial liposomes, along with suitable anchormolecules (such as the biotinated lipids discussed previously). Hereagain, a variety of reconstitution methods, such as the methods of Donget al. (Efficient purification and reconstitution of p-glycoprotein forfunctional and structural studies, J. Biol. Chem. 271 (46) 28875-28883,1996), may be used.

[0180] A P-glycoprotein conformation sensitive antibody, such as UIC2 (amouse antibody), serves as the reagent ligand for this assay. Thisantibody may be either tethered directly to the assay surface, asdescribed previously, or else tethered indirectly by binding to anactive surface tethered anti-antibody (second antibody).

[0181] The liposomes containing the fluorescent-labeled P-glycoproteinsare then applied to the detector surface (which will usually be part ofa flow cell, or flow cell component, such as a capillary tube), andtheir anchor groups are allowed to bind.

[0182] Since the transport ability of P-glycoprotein is ATP dependent,appropriate levels of ATP and magnesium (required for ATP hydrolysis)may also be added to the assay reaction buffer at various times duringthe assy.

[0183] Over the course of the assay, the distribution of theP-glycoprotein molecules will vary. In the absence of P-glycoproteinbinding test ligands, the fluorescently labeled P-glycoprotein moleculeswill not bind to the surface bound, UIC2 like, antibody. They willrandomly diffuse throughout the liposome membrane, and thus produce arelatively small evanescently stimulated fluorescent signal. Bycontrast, in the presence of a P-glycoprotein binding test ligand, thefluorescent-labeled P-glycoprotein molecules will bind to the UIC2 likeantibody, and thus distribute close to the evanescent wave-emittingsurface. This will produce a relatively large evanescently stimulatedfluorescent signal. Test ligands that bind to P-glycoprotein can thus bequickly detected. These test ligands can then be flagged for furtherstudy.

Example 6 Cytochrome P450 Binding Assay

[0184] The evanescent-liposome techniques disclosed here can also beused to study the binding of enzyme substrates to membrane boundenzymes, such as members of the cytochrome P450 family. This family ofmembrane proteins is responsible for the majority of drug breakdown ormetabolism in the body. Different individuals possess different isozymesof cytochrome P450, and thus differ in their ability to metabolize anygiven drug. Thus an important part of ADMET analysis is characterizingthe interaction between a drug candidate, and the various cytochromeP450 enzymes.

[0185] Previous work has shown that cytochrome P450 substrate ligands(type I ligands) may be used to displace cytochrome P450 from surfacebound triazole-based general P450 inhibitors (type-II ligand) (Winteret. al., “A microsomal ecdysone-binding cytochrome P450 from the insectLocusta migratoria purified by sequential use of type-II and type-Iligands” Biol Chem 2001 November; 382(11): 1541-9). In this earlierwork, this displacement was used in affinity columns to purifycytochrome P450 enzymes of interest. However this displacement techniquemay also be utilized for the evanescent liposome methods of the presentinvention.

[0186] Here, cytochrome P450 binding assays may be performed by testingthe ability of an unknown test ligand to displace a given cytochromeP450 target membrane receptor from an evanescent surface bound P450inhibitor, which serves as a reagent ligand. Here, as in the work ofWinter, this reagent ligand may be a triazole-based general P450inhibitor (type-II ligand).

[0187] To do this, as always, the Cytochrome P450 family target membranereceptors need to be labeled. A good way to do this is by fusion withgreen fluorescent protein, following the methods of Rainov et. al. “Achimeric fusion protein of cytochrome CYP4B1 and green fluorescentprotein for detection of pro-drug activating gene delivery and for genetherapy in malignant glioma” Adv Exp Med Biol 1998;451:393-403. Suchhybrid fusion cytochrome P450—green fluorescent protein hybrids havebeen shown to retain their proper spectrum of enzymatic activity.

[0188] Liposome synthesis: fluorescent cytochrome P450, liposome lipids,and appropriate anchor groups, may be incorporated into liposomes usinga variety of methods, including the detergent dialysis methods describedpreviously. Since cytochrome P450 requires activation with cytochromeP450 reductase, and cofactors such as NADPH, NADH, FAD, FNM, etc., thesecofactors may additionally be incorporated into the liposome and/or thereaction buffer as appropriate.

[0189] In the absence of test ligands with cytochrome P450 bindingability, the fluorescent cytochrome P450 target membrane receptors willbind to the surface bound cytochrome P450 inhibitors, and thus exhibit arelatively strong level of evanescent wave stimulated fluorescence. Inthe presence of test ligands with cytochrome P450 binding ability,however, the fluorescent cytochrome P450 target membrane receptors willbecome displaced from the surface bound inhibitor. This will result in adecrease in the amount of evanescent wave stimulated fluorescence.

[0190] Test ligands that bind to various cytochrome P450 family membersof interest can thus be quickly detected. These test ligands can then beflagged for further study.

Example 7 Composite Test Devices

[0191] In many embodiments, it may be desirable to combine one or moretest elements of the present invention with micro-fluidic switchelements. Such micro-fluidic switches can be used to dynamicallyredirect the passage of a test ligand through a multiple of differentdetector elements, depending upon the results of earlier tests in theseries. Usually, the micro-fluidic switches and detection elements willbe computer controlled.

[0192] As an example, consider a multi-element device consisting of aseries of target receptor binding elements, a series of non-targetreceptor elements, a series of ADMET detector elements, and ahigh-performance, but low throughput, test ligand analyzer such as amass spectrometer. Here, micro fluidic switches may direct the passageof the test ligand through the system. As an example, depending upon theresults of the binding element portion of the system, a non-targetbinding test ligand may be directed to a waste container, and subsequentnon-target and ADMET analysis skipped. Those test ligands withappropriate target and non-target binding properties may be directed tothe ADMET section. In turn, those test ligands with appropriate ADMETcharacteristics may be directed to a general purpose, high-performance,test ligand analytical system, such as the mass spectrometer examplegiven previously. This way, promising test ligands may be almostinstantly identified and characterized.

[0193] Such micro fluidic-switched, multi-element methods, may also bedesirable for speeding up test throughput. Typically, a set of unknowntest ligands will only contain a few members with the ability to bind tothe desired set of target membrane receptors. Since usually, no furtheranalysis of test ligands without the desired binding capability isnecessary, further analysis may be skipped and these test ligands may bediscarded. Here, micro fluidic switching elements enable the creation ofnetworked multi-element drug detection flow cell devices. Here, a largenumber of test ligands is screened in many different reaction cells forthe desired reactivity to appropriate target membrane receptors. Thosethat are seen to have the appropriate binding capability are thendirected by micro fluidic switches to a smaller number of reactioncells, where a second stage of binding to non-target membrane receptorsis assessed. Those few test ligands that pass this second stage test arethen directed by micro fluidic switches to a relatively small number ofADMET flow cell sensors. At any point in the analysis, interesting testligands can be diverted to a general purpose high performance ligandanalyzer, such as a mass spectrometer.

[0194] In this way, a high-performance micro-analytical drug discoverysystem may be created. Such a system could work using ultra smallquantities of materials, such as the material created from a singlecombinatorial synthesis bead, and could significantly reduce the amountof materials, time, and effort required for the drug discovery process.

Example 8 Composite Test Supports

[0195] As previously discussed, use of composite support materials maybe desirable in some cases. FIG. 11 shows an example of such a compositesupport. An underlying flat energy emitting material (1), such as glassor plastic, that has an index of refraction significantly different fromthe assay's aqueous media, is covered with a variable thickness coatingof a different transmission material (2), such as a water permeableporous polymer, that has an index of refraction substantially similar tothe aqueous media. In this example, the different transmission material(2) has been deposited in a configuration that creates a variabledistance from the underlying support material (1) by replica molding,photolithography, or other process. Evanescent waves coming from theinterface between energy emitting material (1) and transmission material(2) thus are attenuated to a variable extent depending upon the localthickness of transmission material (2). If transmission material (2) ischosen to have an index of refraction similar to the assay's aqueousmedia outside of material (2), this evanescent wave will continue topropagate through material (2) and the boundary between material (2) andthe assay's aqueous media in an essentially undisturbed manner. As aresult, the intensity of the evanescent wave in the assay's aqueousregion outside of material (2) will vary as a function of material (2)'sthickness.

[0196] In some cases, it may be useful to modify transmission material(2) to enable it to support (and irreversibly anchor) lipid bilayers byusing silane-polyethyleneglycol-lipid as a cushion and covalent linkerlayer (3), following the methods of Wagner and Tamm (TetheredPolymer-Supported Planar Lipid Bilayers for Reconstitution of IntegralMembrane Proteins: Silane-Polyethyleneglycol-Lipid as a Cushion andCovalent Linker, Biophysical Journal Volume 79 September 20001400-1414), or equivalent methods. The net effect is to produce a bumpysupport surface (2,3) that anchors a lipid bilayer at a variabledistance from the underlying energy-emitting surface (1). This variableheight lipid bilayer (4) contains laterally mobile target membranereceptors (5, 6), which are labeled with reporter groups. Because theyare laterally mobile, these target membrane receptors can freely diffuseto various locations far away from (5) or close to (6) theenergy-emitting surface (1).

[0197] The composite test support may further contain regions wheretethered reagent ligands are bound (7). A replica molding,photolithographic, or other process may be used to create such regions.Ideally such tethered reagent-ligand regions (7) will be located eitherfar away from, or close to, energy-emitting surface (1), so as tomaximize the signal difference between the reagent-ligand bound (6) andunbound (5) state of the target membrane receptors. The net effect is tocreate the same type of membrane receptor assay as discussed previously,but with an alternate geometry that does not necessarily require the useof liposomes.

Example 9 Drug Manufacturing

[0198] The membrane receptor reagent and assay disclosed here also hasutility for drug manufacturing. This is particularly useful forbiotherapeutic drugs. Many biotherapeutic drugs function by interactingwith specific target membrane receptors. In contrast to traditionalpharmaceuticals, which typically are comparatively small (low molecularweight) molecules with well-defined structures, biotherapeutics aretypically larger molecules, such as proteins, which have a more complexstructure that is less well defined. Due to their complex structure,protein based biotherapeutic drugs can be produced with variable levelsof posttranslational modification, and in various conformations. Suchposttranslational modification and conformational states can vary as aresult of subtle changes in the manufacturing process.

[0199] Many of these subtle posttranslational modifications have littleeffect on the biotherapeutic drug's overall charge or mass, and thus aredifficult to detect by many commonly used analytical methods. Forexample, subtle changes, such differences between glycosylation on afirst amino acid residue at a first location along the protein's peptidechain, versus glycosylation on a second amino acid residue (of the sametype) at a second location along the protein's peptide chain, makelittle or no difference to the protein's overall mass, charge, orhydrophilic/hydrophobic balance. Thus these changes can be difficult todetect by conventional capillary electrophoresis, mass spectrometry, or2D gel electrophoresis techniques. Such changes, however, can be veryimportant because they can affect the binding specificity of thebiotherapeutic drug, causing diminished biding to the drug's intendedtarget membrane receptor, or enhanced binding to non-target membranereceptors, resulting in unwanted side effects.

[0200] Membrane receptor assays can be constructed that are sensitive tosuch minor structural variations, however. For example, a membranereceptor assay may be constructed that contains both the primary targetmembrane receptor for a properly formed biotherapeutic drug, and variousnon-target membrane receptors where defective forms of the drug mightbind. The output from a manufacturing process (such as a cell cultureapparatus, separation apparatus, etc.) may be monitored by amulti-component membrane test apparatus (such as discussed in example 7and FIG. 10), and deviations from ideal production quickly detected andcorrected.

[0201] An additional advantage is that the methods of the presentdisclosure also tend to directly disclose the biological significance ofsuch posttranslational modification differences. If, for example, amisplaced glycosylation reaction results in a decreased binding to theappropriate target membrane receptor, or increased binding to aninappropriate non-target membrane receptor, the biological significanceof the change is immediately evident. By contrast, traditionalanalytical methods give little clue as to the biological significance ofsuch differences.

[0202] Thus the methods of the present disclosure can be quite usefulfor manufacturing process control and/or quality control purposes. Thiscan help manufacturers improve yields and reduce the chances ofinadvertently releasing bad products. Additionally, these methods canhelp manufacturers distinguish between various different types ofmanufacturing processes, and help select the manufacturing process thatmost consistently produces the desired form of the drug product.

[0203] The methods of the present disclosure can also help amanufacturer optimize the structure of a drug for most consistentmanufacturability. For example, if the membrane receptor assay detectsthat a particular amino acid region along a drug's polypeptide chain hasa history of problematic posttranslational variation, a manufacturer maychose to optimize this amino acid region. Again using the example ofvariable glycosylation between a first and a second amino acid residue,the manufacturer may chose to solve the problem by changing thestructure of the drug to eliminate or alter the second amino acidresidue.

1. A membrane-receptor reagent for measuring the interaction betweentest analytes and target membrane receptors, comprising; a supportcontaining an energy-emitting surface that emits energy that varies as afunction of distance from said surface; tethers associated with saidsupport; reagent ligands attached to said tethers; a fluid lipidmembrane in the form of a liposome, phospholipid vesicle, or lipidbilayer mounted on a second surface that projects away from said energyemitting surface, comprising target membrane receptors that bind totarget analytes and reversibly bind to said reagent ligands, whereinsaid target membrane receptors move fluidly within said fluid lipidmembrane and further comprise integral, peripheral, or transmembraneproteins labeled with a moiety that produces a detectable signal uponreceiving excitation energy; and anchoring means for irreversiblybinding said fluid lipid membrane to said support; wherein bindingbetween said target membrane receptors and target analyte either blocksor promotes binding between said reagent ligands and said targetmembrane receptors, causing a change in an average distance between saidtarget membrane receptors and said energy emitting surface, resulting ina change in said detectable signal.
 2. The reagent of claim 1, in whichsaid energy emitted by said surface is selected from the groupconsisting of evanescent waves, surface plasmon resonance, and electrontransfer, and the detectable signal is selected from the groupconsisting of fluorescence, luminescence, electrochemiluminescence,electron transport, and surface plasmon resonance.
 3. The reagent ofclaim 1, in which said label moiety is located on an integral orperipheral membrane protein that binds to the target membrane receptorduring at least one of the various target membrane receptorconformational states.
 4. The reagent of claim 1, wherein said anchormeans is selected from the group consisting of protein-ligand bindingreagents, electrostatic interactions, hydrophobic interactions,polymeric tethers, and polymeric meshes.
 5. The reagent of claim 1,wherein said target membrane receptors are membrane proteins selectedfrom the group consisting of 7-transmembrane proteins, G-protein coupledreceptors (GPCR), toll receptors, ion channels, ABC cassette pumps,hormone receptors, biological response modifier receptors, apoptosisreceptors, angiogenesis receptors, neuroreceptors, histocompatiblityantigens, coagulation factors, immune response antigens, and cytochromeP450 enzymes.
 6. The reagent of claim 1, wherein said target membranereceptors and test analytes are selected from membrane receptors andanalytes used in drug discovery, absorption distribution, metabolism,and excretion (ADME), toxicology, cellular proliferation, cellularregulation, and medical diagnostics assays.
 7. The reagent of claim 1,further comprising a reference energy source, which generates anormalization signal representative of the number of said signalgenerating label moieties.
 8. The reagent of claim 1, wherein saidsupport is a microarray, capillary tube, fiber optic fiber, or flow celldevice.
 9. A method for measuring the interaction between test analytesand target membrane receptors, comprising the steps of: a) providing amembrane receptor reagent comprising: a support containing anenergy-emitting surface that emits energy that varies as a function ofdistance from said surface; tethers associated with said support;reagent ligands attached to said tethers; a fluid lipid membrane in theform of a liposome, phospholipid vesicle, or lipid bilayer mounted on asecond surface that projects away from said energy emitting surface,comprising target membrane receptors that bind to target analytes andreversibly bind to said reagent ligands, wherein said target membranereceptors move fluidly within said fluid lipid membrane and furthercomprise integral, peripheral, or transmembrane membrane proteinslabeled with a moiety that produces a detectable signal upon receivingexcitation energy; and anchoring means for irreversibly binding saidfluid lipid membrane to said support; b) measuring an initial detectablesignal emitted from the signal generating label moieties in saidreagent; c) adding a sample comprising target analytes to saidmembrane-receptor reagent, wherein binding between said target membranereceptors and target analyte either blocks or promotes binding betweensaid reagent ligands and said target membrane receptors, causing achange in the average distance between the target membrane receptors andthe energy emitting surface, resulting in a change in the detectablesignal; and d) measuring changes in said detectable signal after samplehas been added, wherein said changes in said detectable signal indicatesthe interaction between said target analyte and said target membranereceptors.
 10. The method of claim 9, in which said label moiety islocated on an integral or peripheral membrane protein that binds to thetarget membrane receptor during at least one of the various targetmembrane receptor conformational states.
 11. The method of claim 9, usedas a method of manufacturing a drug compound, in which the test analytesare candidate drug molecules, and the method is used to optimize thedrug structure or manufacturing means.
 12. The method of claim 9,wherein said energy-emitting surface emits evanescent waves, and saiddetectable signal is a fluorescent or luminescent signal.
 13. The methodof claim 9, in which the changes in the detectable signal are adjustedby a reference signal.
 14. The method of claim 9, where said support isa microarray, capillary tube, fiber optic fiber, or flow cell device.15. The method of claim 9, wherein said target membrane receptors andtest analytes are selected from membrane receptors and analytes used indrug discovery, absorption distribution metabolism and excretion (ADME),toxicology, cellular proliferation, cellular regulation, and medicaldiagnostics assays.
 16. The method of claim 9, wherein fluidic switchelements dynamically direct the passage of the test analytes todifferent test devices or regions of the test device.
 17. A method formaking a drug compound, wherein the drug structure is identified oroptimized by the steps of: a) providing a membrane-receptor reagentcomprising: a support containing an energy-emitting surface that emitsenergy that varies as a function of distance from said surface; tethersassociated with said support; reagent ligands attached to said tethers;a fluid lipid membrane in the form of a liposome, phospholipid vesicle,or lipid bilayer mounted on a second surface that projects away fromsaid energy emitting surface, comprising target membrane receptors thatbind to target drug molecules and reversibly bind to said reagentligands, wherein said target membrane receptors move fluidly within saidfluid lipid membrane and further comprise integral, peripheral, ortransmembrane membrane proteins labeled with a moiety that produces adetectable signal upon receiving excitation energy, and anchoring meansfor irreversibly binding said fluid lipid membrane to said support; b)measuring an initial detectable signal emitted from the signalgenerating label moieties in said reagent; c) adding a sample comprisingtarget drug molecules to said membrane-receptor reagent, wherein bindingbetween said target membrane receptors and target analyte either blocksor promotes binding between said reagent ligands and target membranereceptors, causing a change in the average distance between the targetmembrane receptors and the energy-emitting surface, resulting in achange in the detectable signal; and d) measuring changes in saiddetectable signal after sample has been added, wherein said changes insaid detectable signal indicates the amount of interaction between saidtarget drug molecules and said target membrane receptors.
 18. The methodof claim 17, in which the drug molecules are proteins, and the changesin the detectable signal represent different posttranslationalmodification states or different conformational states of the drugcompound.
 19. The method of claim 17, in which the drug molecules areproteins, and the changes in the detectable signal represent differentposttranslational modification states or different conformational statesof the drug compound, and the method is used for manufacturing processcontrol or quality control purposes.
 20. The method of claim 17, inwhich the drug molecules are manufactured by a process that produces avariable mixture of different molecular forms of the drug, and themethod is used for manufacturing process control or quality controlpurposes.